Amylase activity in different temperatures

Apparatus

- 0.5% starch buffered to pH 6.7

- 1% NaCl solution

- 5% Fearon’s reagent (methylamine hydrochloride)

- 20% NaOH

- pH 6.7 buffer

- drinking/distilled water

- boiling tubes

- test tube rack

- 100 ml measuring cylinder

- 1 ml, 5 ml and 10 ml pipettes and pumps to fit

- stop clock

- colorimeter

- plastic cups

- water baths

Method

"As per the laboratory script"

Results

Evaluation

The results show that the activity of amylase is very slow at 4oC, this is due to the enzyme and substrate having less kinetic energy thus not being able to form a lot of enzyme-substrate complexes. The graph shows a steep rise in absorbance between 4oC and 40oC, this shows that as the temperature increased so did the rate of hydrolysis of starch into maltose. As the temperature increases the molecules vibrate more due to more kinetic energy, this speeds up the activity of the amylase as the substrates move faster and there is a higher chance of successful collisions. The graph shows that between 50oC to 70oC there is a steep decrease in absorbance meaning there has been fewer hydrolysis of starch. The decrease in absorbance means that the amylase enzyme became less active. Enzymes can only withstand certain temperatures, the graph shows that the optimum temperature of amylase is 40oC as that is the temperature where the hydrolysis of starch to maltose was the fastest. The decrease of activity of the enzyme amylase after 40oC was due to the enzyme having too much kinetic energy. Instead of increasing the reaction rate, the molecules start to vibrate at more rapid speeds and thus the weak hydrogen bonds that hold the protein start to break. Breaking of hydrogen bonds changes the shape of the enzyme thus altering the active site hence no substrate can bind to it. No enzyme-substrate complex can be formed so the starch can not be broken down to maltose. The enzyme has been denatured.

Even though this experiment was reliable and done as accurately as possible there were still some limitations to how accurately this type of experiment can be done. Some of the errors or limitations were due to the apparatus and some are human errors. The experiment can be more reliable if each there were 3 mixtures used in separate boiling tubes for each temperature. An average can be taken from the 3 results for each temperature making the experiment more reliable. The methods shows to do the experiment with all the different temperatures with a 30second time gap. This can make the experiment unreliable as the boiling tubes can be placed a little earlier or later than the allocated 30 seconds. More reliability of the experiment can be maintained if each different temperature is done after the previous has finished, this may take longer but will give more reliable results.